Characterization of Two AGAMOUS-like Genes and Their Promoters from the Cymbidium faberi (Orchidaceae)

Arabidopsis AGAMOUS (AG) play roles in determining stamens’ and carpels’ identities, floral meristem determinacy, and repression of the A-function. Gynostemium fused by stamens and carpels is a characteristic reproductive structure in orchid flowers, which shows a considerable difference from the reproductive organs of eudicots and other monocot species. The molecular basis of orchid gynostemium development remains largely unknown. Here, we report the identification and functional characterization of two AG-like genes, CyfaAG1 and CyfaAG2, and their promoters from C. faberi. Both CyfaAG1 and CyfaAG2 are highly expressed in the anther cap, gynostemium, and ovary. Ectopic expression of CyfaAG1 and CyfaAG2 promotes early flowering of wild-type Arabidopsis. Moreover, ectopic expression of CyfaAG1 completely rescues floral defects in the Arabidopsis ag-1 mutant, while ectopic expression of CyfaAG2 only completes filament and carpel development. Our findings suggest that CyfaAG1 acts as an evolutionarily conserved C-function gene in determining reproductive organ identity and mediating floral meristem determinacy. CyfaAG2 redundantly mediates the C-function in floral meristem determinacy and gynostemium development. Our results provided more details to understand how the C-class function has been partitioned in orchids, and the roles of two AG orthologs in regulating gynostemium development in C. faberi.


Introduction
Cymbidium faberi Rolfe is a very popular potted orchid and has been cultivated for centuries in China, Japan, and Korea [1]. As a Chinese traditional famous flower, numerous varieties with diverse flower phenotypes have been developed during several thousand years of domestication. However, the mechanism for regulating the floral organ morphogenesis of C. faberi remains unclear. Previous studies suggested that different homeotic MADS transcription factors work together to specify the identities of different types of floral organs during flower development [2,3]. In Arabidopsis, the C-class MADS-box transcription factor AGAMOUS (AG) plays a key role in regulating stamen and carpel identities as well as meristem determinacy [4]. Moreover, most AG orthologs from other eudicots retain functional conservation in determining reproductive organ identity and meristem determinacy during flower development [5]. However, the C-class function has been partitioned among monocots, since the AG lineage is further duplicated within this taxa [6][7][8][9]. In rice, two C-class genes, OsMADS3 and OsMADS58, have diversified with separate functions: OsMADS3 is crucial for stamen identity and OSMADS58 is crucial for floral meristem determinacy and carpel morphogenesis [6,10]. Moreover, both genes share redundancy in the regulation of stamen and carpel identities [6,11,12]. The C-class function has also been partitioned in maize, ZMM2 and ZMM23 are orthologs of OsMADS3 and are crucial for organ (stamen and carpel) identity, while ZAG1 is an ortholog of OsMADS58 and OsMADS58 and crucial for floral meristem determinacy [13,14]. In orchids, the C-class gene DOAG1 from Dendrobium was proved to specify reproductive organs, mediate perianth development, and regulate floral meristem determinacy [7]. In C. sinense (Orchidaceae), two AG-like genes CsAG1 and CsAG2 also showed functional redundancy and divergence. CsAG1 expressed in the tepals, lips, and gynostemium, and was involved in reproductive organ development, while CsAG2 was highly expressed in the gynostemium but its function remained unclear [9]. In addition, an AG-like gene, CeMADS1, may have a pivotal C function in reproductive organ development in C. ensifolium, while the function of another C-class AG-like gene, CeMADS2, remains unclear [15].
The gynostemium, a column of fused stamens and carpels, is a characteristic reproductive structure in the orchid flower, which shows a considerable difference from the reproductive organ of eudicots and other monocot species. Moreover, two AG-like genes resulted from a duplication event predating the divergence of Orchidaceae species [9]. Although one AG lineage gene harboring C function was identified in Dendrobium, C. sinense, and C. ensifolium [7,9,15], the function of another AG lineage gene should be further extensively explored. In this study, we isolated two C-class genes, CyfaAG1 and Cy-faAG2, and their promoters from C. faberi. The flower of C. faberi consists of three whorls of floral organs, with three sepals in whorl 1, two petals and a lip in whorl 2, and gynostemium-fused stamens and carpels in whorl 3 ( Figure 1). In addition, we also characterized the functions of both genes and promoters. Our results provided more details to understand how C-class function has been partitioned in orchid, and the functions of two AG-like genes involved in the gynostemium development of C. faberi.   faberi; (C) anther cap, gynostemium, and ovary of C. faberi. Sepals (sep), petals (pet), lips (lip), anther cap (anc), gynostemium (gyn), ovary (ova).  (Figure 2), and the genes were designated as CyfaAG1 (Cymbidium faberi AGAMOUS) and CyfaAG2, respectively.

Deletion Analysis of pCyfaAG1 and pCyfaAG2 in Transgenic Arabidopsis
A GUS reporter gene separately driven by a series of 5 deletions fragments of pCy-faAG1 and pCyfaAG2 was assayed in transgenic Arabidopsis to analyze the regulatory effect of different regions of the corresponding promoter (Figures 3-5). GUS staining was separately examined in the T1 generation of different deletion constructs. Moreover, GUS staining was obviously observed in the inflorescence and mature flower where sepal, stamen (filament and anther), stigma, and stigmatic papillae staining were intense, but was almost absent in petal of p1D1::GUS, p1D2::GUS, and p1D3::GUS transgenic Arabidopsis ( Figure 3D,F,G,I,J,L). However, GUS staining suggested that pCyfaAG1 drove GUS to extensively express in the sepal, filament, and gynoecium of stage 12 floral buds, but GUS expression was not observed in the anther and petal in p1D1::GUS transgenic Arabidopsis ( Figure 3E) [29]. In addition, relatively weak GUS staining was separately observed in the sepal and gynoecium of stage 12 floral buds, but was almost absent in the petal and stamen of the stage 12 floral buds in p1D2::GUS and p1D3::GUS transgenic Arabidopsis ( Figure 3H,K). In addition, GUS staining was obviously observed in the inflorescence and mature flower where sepal, stamen, and gynoecium were intense, but was absent in petal of p2D1::GUS and p2D2::GUS transgenic Arabidopsis, respectively ( Figure 4D,F,G,I). Moreover, GUS staining was also observed in the sepal, filament, and gynoecia of stage 12 floral buds, but was absent in the anther and petal in p2D1::GUS and p2D2::GUS transgenic Arabidopsis, respectively ( Figure 4E,H). However, GUS staining was only found in floral bud from appearance until stage 12 of p2D3::GUS transgenic Arabidopsis ( Figure 4J). Furthermore, GUS staining was observed only in the gynoecium even in stage 12 of p2D3::GUS transgenic Arabidopsis ( Figure 4K). A further deletion of the −956/−51 fragment from p2D2 (−956/+187) to produce p2D3 (−50/+187) caused obviously decreased GUS activity in transgenic Arabidopsis ( Figure 4J,K and Figure 5B). These results suggested that the −956/−51 regions are capable of inducing pCyfaAG2 promoter activity in the sepal, stamen, and gynoecia, and a 1143 bp region (−956/+187) of pCyfaAG2 was sufficient for driving the CyfaAG2 gene to regulate stamen and gynoecium development.

Expression Analysis of CyfaAG1 and CyfaAG2
CyfaAG1 was mainly expressed in root, leaf, anther cap, gynostemium, and ovary, while CyfaAG2 showed relatively narrower expression zones and was expressed only in the anther cap, gynostemium, and ovary of C. faberi ( Figure 6A). In addition, the expression level of CyfaAG1 in the gynostemium was separately significantly higher than in the root, leaf, anther cap, and ovary (p < 0.05), while the expression level of CyfaAG2 in the anther cap was significantly higher than in the gynostemium and ovary (LSD, p < 0.05) ( Figure 4A). Moreover, the expression level of CyfaAG2 in the anther cap or gynostemium was significantly higher than that of CyfaAG1 in the anther cap or gynostemium, respectively (p < 0.05) ( Figure 6A). CyfaAG1 and CyfaAG2 transcripts became detectable after primodium emergence of floral buds ( Figures 6B and 7A). During the seasonal floral bud dormancy by winter chilling (S2), both genes were expressed at low levels ( Figure 4B). Moreover, with the dormancy broken and microspore mother cell beginning meiosis, the expressions of CyfaAG1 and CyfaAG2 increased obviously ( Figures 6B and 7E,F). Furthermore, CyfaAG1 expression reached a peak at monokaryotic microspore within tetrads (S6), while CyfaAG2 expression reached a peak at 2-cell pollen within mature tetrads of pollinium at the first day of flower opening ( Figures 6B and 7N,O,Q,R). However, CyfaAG2 expression was separately significantly higher than that of CyfaAG1 in S3 and of the firstday opening flower (LSD, p < 0.05) ( Figure 6B).

Expression Analysis of CyfaAG1 and CyfaAG2
CyfaAG1 was mainly expressed in root, leaf, anther cap, gynostemium, and ovary, while CyfaAG2 showed relatively narrower expression zones and was expressed only in the anther cap, gynostemium, and ovary of C. faberi ( Figure 6A). In addition, the expression level of CyfaAG1 in the gynostemium was separately significantly higher than in the root, leaf, anther cap, and ovary (p < 0.05), while the expression level of CyfaAG2 in the anther cap was significantly higher than in the gynostemium and ovary (LSD, p < 0.05) ( Figure 4A). Moreover, the expression level of CyfaAG2 in the anther cap or gynostemium was significantly higher than that of CyfaAG1 in the anther cap or gynostemium, respectively (p < 0.05) ( Figure 6A). CyfaAG1 and CyfaAG2 transcripts became detectable after primodium emergence of floral buds ( Figures 6B and 7A). During the seasonal floral bud dormancy by winter chilling (S2), both genes were expressed at low levels ( Figure 4B). Moreover, with the dormancy broken and microspore mother cell beginning meiosis, the expressions of CyfaAG1 and CyfaAG2 increased obviously ( Figures 6B and 7E,F). Furthermore, CyfaAG1 expression reached a peak at monokaryotic microspore within tetrads (S6), while CyfaAG2 expression reached a peak at 2-cell pollen within mature tetrads of pollinium at the first day of flower opening ( Figures 6B and 7N,O,Q,R). However, CyfaAG2 expression was separately significantly higher than that of CyfaAG1 in S3 and of the first-day opening flower (LSD, p < 0.05) ( Figure 6B).

Expression Analysis of CyfaAG1 and CyfaAG2
CyfaAG1 was mainly expressed in root, leaf, anther cap, gynostemium, and ovary, while CyfaAG2 showed relatively narrower expression zones and was expressed only in the anther cap, gynostemium, and ovary of C. faberi ( Figure 6A). In addition, the expression level of CyfaAG1 in the gynostemium was separately significantly higher than in the root, leaf, anther cap, and ovary (p < 0.05), while the expression level of CyfaAG2 in the anther cap was significantly higher than in the gynostemium and ovary (LSD, p < 0.05) ( Figure 4A). Moreover, the expression level of CyfaAG2 in the anther cap or gynostemium was significantly higher than that of CyfaAG1 in the anther cap or gynostemium, respectively (p < 0.05) ( Figure 6A). CyfaAG1 and CyfaAG2 transcripts became detectable after primodium emergence of floral buds ( Figures 6B and 7A). During the seasonal floral bud dormancy by winter chilling (S2), both genes were expressed at low levels ( Figure 4B). Moreover, with the dormancy broken and microspore mother cell beginning meiosis, the expressions of CyfaAG1 and CyfaAG2 increased obviously ( Figures 6B and 7E,F). Furthermore, CyfaAG1 expression reached a peak at monokaryotic microspore within tetrads (S6), while CyfaAG2 expression reached a peak at 2-cell pollen within mature tetrads of pollinium at the first day of flower opening ( Figures 6B and 7N,O,Q,R). However, CyfaAG2 expression was separately significantly higher than that of CyfaAG1 in S3 and of the firstday opening flower (LSD, p < 0.05) ( Figure 6B).

Ectopic Expression of CyfaAG1 and CyfaAG2 in Arabidopsis ag-1 Mutant
To further evaluate the functional divergence of CyfaAG1 and CyfaAG2, we attempted to rescue the loss-of-function Arabidopsis ag-1 mutant using CyfaAG1 and CyfaAG2. 35S::CyfaAG1 and 35S::CyfaAG2 constructs were separately introduced into heterozygote AG/ag-1 Arabidopsis to create complementation lines by agrobacterium-mediated transformation. Transgenic Arabidopsis mutant-allele lines were identified by dCAPS genotyping (Supplementary Figure S3) and were further confirmed by qRT−PCR. Moreover, sixteen 35S::CyfaAG1 lines under wild-type background and 13 independent 35S::CyfaAG1 lines under homozygous ag-1 mutant background were obtained, while ten 35S::CyfaAG2 lines under wild-type background and six independent 35S::CyfaAG2 lines under homozygous ag-1 mutant background were obtained. Furthermore, phenotypes of transgenic Arabidopsis lines were assayed in wild-type and homozygous ag-1 mutant backgrounds to evaluate whether CyfaAG1 and CyfaAG2 could substitute for the endogenous AG gene in determining stamen and carpel identities, respectively. Either 35S::CyfaAG1 or 35S::CyfaAG2 transgenic Arabidopsis under a wild-type background were observed obviously flowering early (Figure 8). spore within tetrads; (P) ovule, enlargement of (N), meiosis of megaspore mother cell; (Q) anther, longitudinal section of the first-day opening flower; (R) pollinium, enlargement of (Q), 2-cell pollen within mature tetrads of pollinium; (S) ovule, enlargement of (Q), linear tetrad (Tet) of megaspores, functional megaspore (Fme) formation.

Ectopic Expression of CyfaAG1 and CyfaAG2 in Arabidopsis ag-1 Mutant
To further evaluate the functional divergence of CyfaAG1 and CyfaAG2, we attempted to rescue the loss-of-function Arabidopsis ag-1 mutant using CyfaAG1 and CyfaAG2. 35S::CyfaAG1 and 35S::CyfaAG2 constructs were separately introduced into heterozygote AG/ag-1 Arabidopsis to create complementation lines by agrobacterium-mediated transformation. Transgenic Arabidopsis mutant-allele lines were identified by dCAPS genotyping (Supplementary Figure S3) and were further confirmed by qRT−PCR. Moreover, sixteen 35S::CyfaAG1 lines under wild-type background and 13 independent 35S::CyfaAG1 lines under homozygous ag-1 mutant background were obtained, while ten 35S::CyfaAG2 lines under wild-type background and six independent 35S::CyfaAG2 lines under homozygous ag-1 mutant background were obtained. Furthermore, phenotypes of transgenic Arabidopsis lines were assayed in wild-type and homozygous ag-1 mutant backgrounds to evaluate whether CyfaAG1 and CyfaAG2 could substitute for the endogenous AG gene in determining stamen and carpel identities, respectively. Either 35S::CyfaAG1 or 35S::CyfaAG2 transgenic Arabidopsis under a wild-type background were observed obviously flowering early (Figure 8).
Among sixteen 35S::CyfaAG1 transgenic Arabidopsis under a wild-type background, one (6.25%) showed a strong phenotype with a sepal converted into a carpeloid organ and a new flower in the center ( Figure 9C,D); two (12.50%) displayed a medium phenotype with a small sepal in whorl 1, stamenoid petal (filament with petal at the top) in whorl 2, and a big pistil in whorl 4 ( Figure 9E,F); four (25.00%) showed a weak phenotype with a small sepal in whorl 1, a small petal in whorl 2, small stamens in whorl 3, and a big pistil in whorl 4 ( Figure 9G,H); and the remaining nine lines (56.25%) had flowers similar to the wild-type Arabidopsis flowers.
Among ten 35S::CyfaAG2 transgenic Arabidopsis under a wild-type background, four (40.00%) produced flowers with a small sepal in whorl 1, a stamenoid petal (filament with petal at the top) in whorl 2, and a big pistil in whorl 4 ( Figure 9I,J); three (30.00%) showed a weak phenotype with a small sepal in whorl 1, a small petal in whorl 2, a small stamen in whorl 3, and a big pistil in whorl 4 ( Figure 9K,L); and the remaining two lines (20.00%) had flowers similar to the wild-type Arabidopsis flowers. Among sixteen 35S::CyfaAG1 transgenic Arabidopsis under a wild-type background, one (6.25%) showed a strong phenotype with a sepal converted into a carpeloid organ and a new flower in the center ( Figure 9C,D); two (12.50%) displayed a medium phenotype with a small sepal in whorl 1, stamenoid petal (filament with petal at the top) in whorl 2, and a big pistil in whorl 4 ( Figure 9E,F); four (25.00%) showed a weak phenotype with a small sepal in whorl 1, a small petal in whorl 2, small stamens in whorl 3, and a big pistil in whorl 4 ( Figure 9G,H); and the remaining nine lines (56.25%) had flowers similar to the wild-type Arabidopsis flowers.  Among thirteen 35S::CyfaAG1 transgenic homozygous ag-1 Arabidopsis, one (7.69%) showed a strong complementation phenotype with a sepal in whorl 1, a petal in whorl 2, a stamen in whorl 3, and a pistil in whorl 4 ( Figure 10C,D); three (23.08%) displayed a medium phenotype with many stamens in whorl 2 ( Figure 10E,F) or sepals in whorl 1, a stamen in whorl 2, and a fat pistil in the interior whorl ( Figure 10E,G); the remaining nine Among ten 35S::CyfaAG2 transgenic Arabidopsis under a wild-type background, four (40.00%) produced flowers with a small sepal in whorl 1, a stamenoid petal (filament with petal at the top) in whorl 2, and a big pistil in whorl 4 ( Figure 9I,J); three (30.00%) showed a weak phenotype with a small sepal in whorl 1, a small petal in whorl 2, a small stamen in whorl 3, and a big pistil in whorl 4 ( Figure 9K,L); and the remaining two lines (20.00%) had flowers similar to the wild-type Arabidopsis flowers.
Among thirteen 35S::CyfaAG1 transgenic homozygous ag-1 Arabidopsis, one (7.69%) showed a strong complementation phenotype with a sepal in whorl 1, a petal in whorl 2, a stamen in whorl 3, and a pistil in whorl 4 ( Figure 10C,D); three (23.08%) displayed a medium phenotype with many stamens in whorl 2 ( Figure 10E,F) or sepals in whorl 1, a stamen in whorl 2, and a fat pistil in the interior whorl ( Figure 10E  Among six 35S::CyfaAG2 transgenic homozygous ag-1 Arabidopsis, one (16.67%) showed a strong complementation phenotype that consisted of flowers only with sepal and pistil whorls, or filament-like organs and pistil ( Figure 10H), one (16.67%) produced flowers with a sepal in whorl 1, filament-like organs in whorl 2, and a fat pistil in the interior whorl ( Figure 10I), and the remaining four lines (66.67%) displayed no complementation and had flowers similar with the flower of the Arabidopsis ag-1 mutant.

Discussion
Orchid reproductive organs (gynostemium) show dramatic changes in morphology compared to the reproductive organs (stamens and carpels) of other angiosperms. In the model species A. thaliana, the floral homeotic C-class gene AG is responsible for the specification of reproductive organ identity and the control of floral meristem determinacy, as well as the prevention of the misexpression of A-function genes in the reproductive organs [4,5,30]. AG expression starts at stage 3 of flower development in the domains of the floral meristem, where stamen and carpel primordia develop, and its expression remains during all stages of stamen and carpel development [11]. Other rosid species AG orthologs, such as PrseAG from Prunus lannesiana [31], KjAG from Kerria japonica [32] and EjAG from Eriobotrya japonica [33], were mainly expressed in stamens and carpels, and showed a conserved function for reproductive organ identity determination in core eudicot species. A duplication event of the AG lineage in the order Ranunculales, a sister lineage to all other eudicots, results in two functionally redundant but distinguishable AG-lineage members [34,35]. ThtAG1 and ThtAG2 are two AG orthologs from Thalictrum thalictroides (Ranunculaceae); ThtAG1 showed a highly conserved C-function, whereas ThtAG2 determined ovule identity [35]. Moreover, NdAG1 and NdAG2 are AG lineage genes from Nigella damascene (Ranunculaceae); NdAG1 confers C-function specifying stamens and carpels identities, while NdAG2 is involved in regulating carpel development and floral determinacy [36]. Two AG-lineage genes, MawuAG1 and MawuAG2, were also found in the basal angiosperm Magnolia wufengensis. MawuAG1 is mainly expressed in stamen, carpel, ovule, and seed, and shows highly conserved C-function in the determination of stamen, carpel, and ovule identity, while MawuAG2 is mainly expressed in stamen and carpel, and may be only involved in stamen development [37]. All these data suggest that gene duplication led to functional redundancy or sub-functionalization of both AG paralogs in eudicots.
In monocot rice, two AG lineage genes, OsMADS3 and OsMADS58, redundantly mediate the C-function: OsMADS3 plays a predominant role in stamen specification, but OSMADS58 plays a predominant role in floral meristem determinacy and carpel morphogenesis [11]. Three AG lineage genes, namely, ZMM2 and ZMM23 (both orthologs of rice OsMADS3), and ZAG1 (ortholog of rice OsMADS58) were found in Zea mays [13,14]. ZAG1 is expressed early in stamen and carpel primordia, and determines the floral meristem, ZMM2 is mainly expressed in the anthers and participates in regulating the formation of stamens and carpels [38]. In orchid C. sinense, CsAG1 expressed in the tepals, lips, and gynostemium, and conferred C-function in reproductive organ development, while CsAG2 was highly expressed in the gynostemium but its function remained unclear [9]. In addition, two AG-like genes, CeMADS1 and CeMADS2, were also found in C. ensifolium [15]. CeMADS1 was expressed only in the gynostemium and may have a pivotal C function in reproductive organ development, while CeMADS2 was expressed in all floral organs and predominantly in gynostemium and its function remained unclear [15].
Here, we have also isolated two AG-like genes (CyfaAG1 and CyfaAG2) from C. faberi. CyfaAG1 is mainly expressed in root, leaf, anther cap, gynostemium, and ovary, while CyfaAG2 was expressed only in anther cap, gynostemium, and ovary of C. faberi. Moreover, pCyfaAG1 and pCyfaAG2 contain CArG-box for MADS-box transcription factor binding and conserved binding sites (AACAAA-/TTTGTT-motif) for floral homeotic APETALA2 transcription factor recognition [16,19]. In addition, both pCyfaAG1 and pCyfaAG2 could drive GUS to extensively express in stamens and carpels of transgenic Arabidopsis. Furthermore, ectopic expressions of CyfaAG1 and CyfaAG2 separately promote early flowering phenotypes. Moreover, ectopic expression of CyfaAG1 produced flowers with sepal converted into carpeloid organ and a new flower in the center or a stamenoid petal (filament with petal at the top) in whorl 2 in wild-type Arabidopsis, while ectopic expression of CyfaAG2 produced flowers with a stamenoid petal (filament with petal at the top) in whorl 2. Furthermore, complementation of loss-of-function Arabidopsis ag-1 mutant suggested that CyfaAG1 could mimic the endogenous AG gene to specify stamen and carpel identity. However, ectopic expression of CyfaAG2 only completes filament and carpel development in the 35S::CyfaAG2 transgenic Arabidopsis ag-1 mutant. Based on these results, it seems that CyfaAG1 and CyfaAG2 work redundantly. CyfaAG1 may confer C-function specifying stamens and carpels identities, as well as floral meristem determinacy, while CyfaAG2 may only be involved in filament and carpel development. Both genes work together to regulate normal gynostemium and ovary development in C. faberi. Our data provide more details to understand how C-class function has been partitioned in orchid, and the functions of two AG-like genes regulating gynostemium development of C. faberi.

Isolation and Characterization of CyfaAG1 and CyfaAG2 from Cymbidium faberi
Total RNA was extracted from floral buds using an EASYspin Plant RNA Kit (Aidlab China) according to the manufacturer's protocol. The first-strand cDNA of 3 RACE was prepared, and then the 3 end cDNA sequences of CyfaAG1 and CyfaAG2 were separately amplified with gene-specific forward primers 3RGSPAG1F and 3RGSPAG2F (Supplementary Table S1) using the 3-full RACE Core Set Ver. 2.0 kit (TaKaRa, Japan) according to the manufacturer's protocol, but with 45 s annealing at 59 • C. The forward primers design was referenced the sequence of AG orthologs CsAG2 (Genbank accession numbers: MG021185.1) and CsAG1 (Genbank accession numbers: MG021184.1) from orchid's close relative species C. ensifolium. In addition, the 5 end cDNA sequences of CyfaAG1 were amplified through 5 RACE using the 5 RACE System for Rapid Amplification of cDNA Ends (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol and the gene-specific primers 5RAG1GSP1, 5RAG1GSP2, and 5RAG1GSP3 (Supplementary Table S1). The 5 end cDNA sequences of CyfaAG2 were amplified according to the above method, but with the genespecific primers 5RAG2GSP1, 5RAG2GSP2, and 5RAG2GSP3 (Supplementary Table S1). The phylogenetic tree was constructed by using MEGA version 5.05 with the neighborjoining (NJ) method. The NJ tree was constructed with 1000 bootstrap replications. All the C-class and D-class MADS-box transcription factors (TF) with complete sequences were obtained from NCBI Genbank (Supplementary Table S2).

Isolation and Sequence
Analysis of CyfaAG1 and CyfaAG2 Promoters from Cymbidium faberi C. faberi genomic DNA was extracted from juvenile leaves using the CTAB Plant Genomic DNA Rapid Extraction Kit (Aidlab, Beijing, China) according to the manufacturer's protocol. The CyfaAG1 5 flanking regions were amplified from C. faberi genomic DNA using the Genome Walking Kit (TaKaRa, Japan) following the manufacturer's protocol and with gene-specific primers pAG1SP1, pAG1SP2, and pAG1SP3 (Supplementary Table S1) for the first walking sequencing, and with the gene-specific primers pAG1SP4, pAG1SP5, and pAG1SP6 (Supplementary Table S1) for the second walking sequencing. Meanwhile, the CyfaAG2 5 flanking regions were amplified according to the above method, but with the gene-specific primers pAG2SP1, pAG2SP2, and pAG2SP3 (Supplementary Table S1) for the first walking sequencing, and with the gene-specific primers pAG2SP4, pAG2SP5, and pAG2SP6 (Supplementary Table S1) for the second walking sequencing, and the gene-specific primers pAG2SP7, pAG2SP8, and pAG2SP9 (Supplementary Table S1) for the third sequencing. The primers designed for the first walking were separately based on the corresponding gene sequences CyfaAG1 (Genbank accession number: MH917913) and CyfaAG2 (Genbank accession number: MH917914). The putative transcription start sites of CyfaAG1 and CyfaAG2 were searched through 5 RACE according to the above method. The cis-acting elements located at the CyfaAG1 and CyfaAG2 promoter regions were separately searched in the PLACE database [39].

Characterization of pCyfaAG1 and pCyfaAG2 Activity from the 5 Deleted Promoter Fragments in Transgenic Arabidopsis
We separately designed three forward primers (TpCyfaAG1F, TpCyfaAG1F1, and TpCyfaAG1F2) and a reversed primer TpCyfaAG1R (Supplementary Table S1) to amplify 5 -deletion fragments of pCyfaAG1. In addition, we also designed three forward primers (TpCyfaAG2F, TpCyfaAG2F1, and TpCyfaAG2F2) and a reversed primer TpCyfaAG2R (Supplementary Table S1) to amplify the promoter deletions of pCyfaAG2, respectively. Three 5 -deletion fragments of pCyfaAG1 were separately designated as p1D1 (−1207/+185), p1D2 (−1191/+185), and p1D3(−387/+185), and then cloned into the pCAMBIA1300 vector with Xba I and Sac I using the ClonExpress ® Ultra One Step Cloning Kit (Vazyme, Nanjing, China) following the manufacturer's protocol. In addition, three 5 -deletion fragments of pCyfaAG2 were separately designated as p2D1 (−1473/+187), p2D2 (−956/+187), and p2D3 (−50/+187), and then cloned into the pCAMBIA1300 vector by using the above method. All the constructs were separately transformed into A. thaliana Col-0 plants (ecotype Columbia) with the floral-dip method suggested by Clough and Bent [40]. Transgenic Arabidopsis seedlings were selected, cultivated, and prepared for histochemical GUS staining according to Zeng et al. [41]. The expression of the GUS gene controlled by different 5 -deletions of pCyfaAG1 and pCyfaAG2 were also separately confirmed via qRT−PCR in transgenic Arabidopsis with the primers qGUSF and qGUSR (Supplementary Table S1). An amplification fragment of A. thaliana Actin (Genbank accession numbers: AY114679.1) with the primers qActinF and qActinR (Supplementary Table S1) was used as the internal control.

Cytomorphological Examination and Expression Analysis of CyfaAG1 and CyfaAG2
The six stages of floral buds described above and first-day opening flower of C. faberi were separately sampled and fixed in FAA (38% formaldehyde: acetic acid: 70% ethanol = 1:1:18, by vol.). The samples were dehydrated in an ethanol series (1.5 h each), cleared in a xylene series (2 h each), infiltrated with a xylene and paraffin (12 h at 38 • C) series, followed by three changes of 100% molten paraffin at 60 • C (4 h each), then embedded into a paraffin block, which was serially sectioned at a thickness of 8 µm with a Leica RM2235 rotary microtome after two days. Subsequently, the sections were stained with safranin-fast green [42]. The sections were observed under a CAIKON RCK-40C microscope and photomicrographs were subsequently taken.
The total RNA of each sample was extracted according to the above method, but the first-strand cDNA was synthesized for quantitative real-time PCR (qRT-PCR) by using the HiScript ® II Q RT SuperMix for qPCR kit (Vazyme, Nanjing, China) following the manufacturer's protocol. The relative expressions of CyfaAG1 and CyfaAG2 were separately detected in root, juvenile leave, sepal, petal, lip, anther, gynostemium, and ovary of C. faberi plants according to Fei and Liu [1], but with the gene-specific forward primer qCyfaAG1F and reverse primer qCyfaAG1R for CyfaAG1, and with the gene-specific forward primer qCyfaAG2F and reverse primer qCyfaAG2R for CyfaAG2, respectively (Supplementary Table S1). The qRT−PCR was performed with three biological replicates and amplification of C. faberi actin (Genbank accession numbers: JN177719.1) fragment was used as the internal control with the forward primer qCyfaactinF and reverse qCyfaactinR (Supplementary Table S1).

Ectopic Expression of CyfaAG1/CyfaAG2 in Arabidopsis ag-1 Mutant and Function Analysis
Full-length CyfaAG1 cDNAs in the sense orientation were cloned into the pBI121 vector with Xba I and Sma I restriction enzymes, and the forward primer TCyfaAG1F and the reverse primer TCyfaAG1R (Supplementary Table S1) under control of the CaMV35S promoter using the ClonExpress ® Ultra One Step Cloning Kit (Vazyme, Nanjing, China) according to the manufacturer's protocol. Full-length CyfaAG2 cDNAs in the sense orientation were cloned into the pBI121 vector according to the above method, but with the forward primer TCyfaAG2F and the reverse primer TCyfaAG2R (Supplementary Table S1). The 35S::CyfaAG1 and 35S::CyfaAG2 constructs were separately transformed into heterozygous Ag/ag-1 Arabidopsis using the floral-dip method suggested by Clough and Bent [40]. Transgenic Arabidopsis seeds were selected and seedlings were subsequently transplanted into soil for cultivation in a growth chamber according to Li et al. [43]. Wild-type, heterozygous AG/ag-1 and homozygous ag-1 transgenic Arabidopsis lines were confirmed by the dCAPS finder program suggested by Neff et al. [44]. The phenotypes of transgenic Arabidopsis were analyzed after flowering. In addition, the complementation degrees of independent transgenic lines of 35S::CyfaAG1 and 35S::CyfaAG2 homozygous ag-1 Arabidopsis were categorized as 'no complementation', 'weak complementation', 'medium complementation' and 'strong complementation', respectively. Moreover, independent transgenic lines of each complementation degree were confirmed by qRT−PCR with the primers qCyfaAG1F and qCyfaAG1R for CyfaAG1 (Supplementary Table S1), and with the primers qCyfaAG2F and qCyfaAG2R suggested above for CyfaAG2 (Supplementary Table S1), respectively. An amplification fragment of A. thaliana Actin (Genbank accession numbers: AY114679.1) with the primers qActinF and qActinR was used as the internal control.

Conclusions
Orchid reproductive organs (gynostemium) show dramatic changes in morphology compared to reproductive organs (stamens and carpels) of other angiosperms. In Arabidopsis, AGAMOUS (AG) play roles in determining stamens and carpels identities, floral meristem determinacy, and repression of the A-function. However, the molecular basis of orchid gynostemium development remains largely unknown. In this study, two AG-like genes, CyfaAG1 and CyfaAG2, and their promoters were isolated and functionally characterized in a Chinese famous traditional orchid C. faberi. Both CyfaAG1 and CyfaAG2 are highly expressed in the anther cap, gynostemium, and ovary. Moreover, both pCyfaAG1 and pCyfaAG2 could drive GUS to extensively express in stamens and carpels of transgenic Arabidopsis. Ectopic expression of CyfaAG1 and CyfaAG2 promotes early flowering of wild-type Arabidopsis. Moreover, ectopic expression of CyfaAG1 completely rescues floral defects in the Arabidopsis ag-1 mutant, while ectopic expression of CyfaAG2 only completes filament and carpel development. Our findings suggest that CyfaAG1 acts as an evolutionarily conserved C-function gene in determining reproductive organ identity and mediating floral meristem determinacy. CyfaAG2 redundantly mediates the C-function in floral meristem determinacy and gynostemium development. Our results provide more details to understand how the C-class function has been partitioned in orchid, and the roles of two AG orthologs involved in the gynostemium development of C. faberi.
Author Contributions: J.L., writing-original draft preparation; L.W., X.C., L.Z. and Y.S., methodology; Z.L., writing-review and editing, supervision. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.